Fish Oil Ameliorates Parenteral Nutrition Associated Liver Disease By IRE1?/XBP1s/JNK Signal Pathway In A Rat Model

Objective:By establishing parenteral nutrition associated-liver disease(PNALD)model in rats,we aim to research the dynamic expression of GRP78,and the related molecules IRE1??p-IRE1? ? XBP1 s ? JNK ? p-JNK in the IRE1 ?/XBP1s/JNK signaling pathway of endoplasmic reticulum stress(ERS)to further explore the role of ERS signal pathway in the pathogenesis of PNALD,and reveal the possible protective molecular mechanism of fish oil-based lipid emulsions in PNALD model,and provide new ideas and basis for clinical prevention and treatment on PNALD.Methods:72 Sprague-Dawley(SD)rats aged 5-6 weeks,body mass 180-220 g,male,were randomly divided into four groups: control group(CON,n=18),normal saline group(NS,n=18),TPN group(PN,n=18),fish oil prevention group(FO,n=18).Parenteral nutrition based on Soybean Oil Fat Emulsion?parenteral nutrition based on ?-3 Fish Oil Fat Emulsion? normal saline were respectively supplied to PN group?FO group?NS group through a PICC catheter inserted in the right jugular vein for one week.The infusion speed that based on weight was 100ml/kg.d on NO.1d,200ml/kg.d on NO.2d,and 300ml/kg.d from NO.3d to NO.7d..CON group and NS group rats were fed standard rat chow and had access to water freely.NO rat chow and water were supplied to PN group and FO group rats.All rats were housed in metabolic cages and subjected to all acclimatization period of a week.Rats were maintained under a 12-hr light-dark cycle at a temperature of 25 ?and a relative humidity of 40%to 60%.Six rats selected from each group randomly were decapitated respectively on 3d,5d,7d in four groups.Serum biochemistry indicators were tested.Liver histological examitations of four groups were observed by HE staining.Expression of GRP78?IRE1??p-IRE1??XBP1s?JNK?p-JNK proteins were detected by Western-blot technique and the expression level of GRP78?IRE1??XBP1s?JNK mRNA gene by reverse transcription-polymerase chain reaction(RT-PCR).Results:1.Comparision of henatoxylin-eosin staining in the liver of four groups:Compared to CON group and NS group,the liver of PN group was mild hepatic steatosis and inflammation cells infiltration on 5d.There were severe hepatic steatosis ?a mass of inflammation cells ?mild hepatic apoptosis and structural distortion in a small number of hepatic cords on 7d of PN group,but no bile duct proliferation or cholestasis.While no obviously bile duct proliferation ?cholestasis?structural distortion?hepatic steatosis or inflammation cells infiltration observed in FO group from 3d to 5d,we only found mild inflammation cells infiltration and hepatic steatosis on 7d.2.The level of serum biochemical indexes: There was significantly difference on the level of AST and ALT among four groups at three times(3d?5d?7d)(P <0.05),and on the level of ALB among four groups at two times(5d?7d)(P <0.05).On the level of TBIL?DBIL?TP?TG?HDL and LDL,there were no differences among four groups(P>0.05).Compared to CON group?NS group and FO group,the level of AST and ALT in PN group was significantly higher on 3d,5d and 7d(P <0.05),while the level of ALB in PN group was lower on 5d and 7d(P <0.05).The level of AST and ALT in PN group and FO group gradually increased with prolonged modeling stimulation.The level of ALB in PN group gradually decreased with prolonged modeling stimulation,while the level of ALB in FO group was no difference at three times.3.The dynamic expression of GRP78?IRE1??p-IRE1??XBP1s?JNK?p-JNK protein of liver tissues in four groups3.1 The dynamic expression of GRP78 protein of liver tissues in four groups: There was significantly difference on the expression of GRP78 protein among four groups at three times(3d?5d?7d)(P <0.05).Compared to CON group and NS group,the expression of GRP78 protein in PN group was higher on 3d,5d and 7d(P<0.05),while the expression of GRP78 protein in FO group was higher merely on 5d and 7d(P <0.05).In contrast to PN group,the expression of GRP78 protein in FO group obviously declined on 3d,5d and 7d(P <0.05).The expression of GRP78 protein in PN group and FO group gradually increased from 5d,and reached peak on 7d(P<0.05).3.2 The dynamic expression of IRE1??p-IRE1? protein of liver tissues in four groups: There was no significant difference in expression of IRE1? protein of liver tissues among four groups or within each group on 3d,5d and 7d(P>0.05),while there was significant difference in the expression of p-IRE1? protein of liver tissues among four groups at three times(P<0.05).Compared to CON group and NS group,the expression of p-IRE1? protein in PN group was higher on 3d,5d and 7d(P<0.05),but the expression of p-IRE1? protein in FO group was higher only on 5d and 7d(P <0.05).In contrast to PN group,the expression of p-IRE1? protein in FO group was obviously lower on 3d,5d and 7d(P <0.05).The expression of p-IRE1? protein in PN group and FO group gradually increased from 5d,and reached peak on 7d(P<0.05).3.3 The dynamic expression of XBP1 s protein of liver tissues in four groups:There was significant difference in expression of XBP1 s protein of liver tissues among four groups at three times(3d?5d?7d)(P<0.05).Compared to CON group and NS group,the expression of XBP1 s protein in PN was higher on 3d,5d and 7d(P<0.05),while the expression of XBP1 s protein in FO group was higher than CON group and NS group only on 5d and 7d(P <0.05).In contrast to PN group,the expression of XBP1 s protein in FO group was obviously lower on 3d,5d and 7d(P <0.05).The expression of XBP1 s protein in PN group reached peak on 5d,but had a downward trend after 5d.The expression of XBP1 s protein in FO group mildly increased from 5d,and reached peak on 7d(P <0.05).3.4 The dynamic expression of JNK ? p-JNK protein of liver tissues in four groups:There was no significant difference in expression of JNK protein of liver tissues among four groups or within each group on 3d,5d and 7d(P>0.05),while there was significant difference in expression of p-JNK protein of liver tissues among four groups at two times(5d?7d)(P<0.05).Compared to CON group and NS group,the expression of p-JNK protein in PN was higher on 5d and 7d(P<0.05).In contrast to PN group,the expression of p-JNK protein in FO group was obviously lower on 3d,5d and 7d(P <0.05).The expression of p-JNK protein in PN group increased from 5d to 7d(P<0.05).4.The dynamic expression of GRP78,IRE1?,XBP1 s,JNK mRNA of liver tissues in four groups4.1 The dynamic expression of GRP78 mRNA of liver tissues in four groups:There was significant difference in expression of GRP78 mRNA of liver tissues among four groups at three times(3d?5d?7d)(P<0.05).Compared to CON group and NS group,the expression of GRP78 mRNA in PN was significantly higher on 3d,5d and 7d(P<0.05),while the expression of GRP78 mRNA in FO group was higher than CON group and NS group merely on 5d and 7d(P <0.05).Compared to PN group,the expression of GRP78 mRNA in FO group was lower on 3d,5d and 7d(P <0.05).The expression of GRP78 mRNA in PN group and FO group increased from 5d to 7d(P<0.05).4.2 The dynamic expression of IRE1? mRNA of liver tissues in four groups:There was significant difference in expression of IRE1? mRNA of liver tissues among four groups at three times(3d?5d?7d)(P<0.05).Compared to CON group and NS group,the expression of IRE1? mRNA in PN group and FO group was significantly higher on 3d,5d and 7d(P<0.05).Compared to PN group,the expression of IRE1? mRNA in FO group was lower on 3d,5d and 7d(P <0.05).The expression of IRE1? mRNA in PN group and FO group increased from 3d to 7d(P<0.05).4.3 The dynamic expression of XBP1 s mRNA of liver tissues in four groups:There was significant difference in the expression of XBP1 s mRNA of liver tissues among four groups at three times(3d?5d?7d)(P<0.05).Compared to CON group and NS group,the expression of XBP1 s mRNA in PN group and FO group was significantly higher on 3d,5d and 7d(P<0.05).Compared to PN group,the expression of XBP1 s mRNA in FO group was lower on 3d,5d and 7d(P <0.05).The expression of XBP1 s mRNA in PN group increased from 3d to 5d,but had a downward trend after 5d(P <0.05).The expression of XBP1 s mRNA in FO group mildly increased from 5d,and reached peak on 7d(P <0.05).4.4 The dynamic expression of JNK mRNA of liver tissues in four groups:There was significant difference in the expression of JNK mRNA of liver tissues among four groups at three times(3d?5d?7d)(P<0.05).Compared to CON group and NS group,the expression of JNK mRNA in PN group was higher on 5d and 7d(P<0.05).While the expression of JNK mRNA in FO group was no obviously difference compared to CON group or NS group(P>0.05).In contrast to PN group,the expression of JNK mRNA in FO group obviously declined on 5d and 7d(P <0.05).The expression of JNK mRNA in PN group increased from 5d to 7d.The expression of JNK mRNA n in FO group was no significantly difference on 3d,5d and 7d(P>0.05).Conclusions:1.In this study,we successfully established PNALD model,and set the foundation for farther studies of its pathogenesis.2.During the process of PNALD model,the expression of the related molecules GRP78,p-IRE1?,XBP1 s,p-JNK protein and IRE1?,XBP1 s,JNK mRNA in the IRE1?/XBP1 s /JNK signaling pathway of ERS increased,and with prolonged modeling stimulation showed a generally progressive increase.It suggests that IRE1?/XBP1 s /JNK signal pathway may participate in PNALD.3.?-3 Fish Oil Fat Emulsion instead of Soybean Oil Fat Emulsion can ameliorate PNALD.We conclude that the protection effect of fish oil may be related to inhibition of IRE1?/XBP1 s /JNK signal pathway.